摘要
目的研究再灌注损伤挽救激酶(reperfusion injury salvage kinase,RISK)信号通路在1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)后适应减轻H9c2心肌细胞缺氧/复氧损伤中的作用。方法将H9c2细胞随机分为7组,即(1)正常对照组(C);(2)缺氧/复氧组(H/R);(3)S1P组;(4)S1P+LY294002组(S1P+LY);(5)LY组;(6)S1P+PD98059组(S1P+PD);(7)PD组。采用MTT法测定各组细胞存活率;比色法测定丙二醛(MDA)含量、总超氧化物歧化酶(TSOD)和锰超氧化物歧化酶(Mn-SOD)活力;采用激光共聚焦显微镜技术检测细胞内游离钙离子浓度变化;流式细胞仪检测心肌细胞凋亡率;Western blot法测定Akt和ERK1/2蛋白的磷酸化水平。结果与H/R组比较,S1P组可明显增加H9c2细胞缺氧/复氧损伤后细胞存活率及凋亡率,增加TSOD及Mn-SOD活力,降低MDA含量,降低钙离子浓度,增加Akt和ERK1/2磷酸化水平,而PI3K/Akt信号通路阻断剂LY294002或ERK1/2阻断剂PD98059可阻断S1P对H9c2的上述作用。结论 S1P能够减轻H9c2细胞缺氧/复氧损伤,加入PI3K/Akt抑制剂LY294002和ERK1/2抑制剂PD98059均使S1P的保护作用被取消,表明S1P通过RISK信号通路发挥抗H9c2细胞缺氧/复氧损伤作用。
Aim To study the protective effects of sphingosine 1-phosphate( S1P) postconditioning on rat myocardial cells injured by hypoxia / reoxygenation in reperfusion injury salvage kinase( RISK) signal pathway. Methods The cultured rat H9c2 cells were randomly divided into seven groups:( 1) control group;( 2) hypoxia /reoxygenation( H/R) group;( 3) S1 P group;( 4) S1 P + LY294002 group( S1 P + LY);( 5)LY group;( 6) S1 P + PD98059 group( S1 P + PD);( 7) PD group. The viability of H9c2 cells was detected using MTT method. The content of MDA in the cultured medium and the activity of T-SOD and MnSOD were measured with colorimetry. The concentration of intracellular free calcium ion was detected by confocal microscopy. The rate of cell apoptosis was determined by flow cytometric analysis. Western blot was used to assess phosphorylation of Akt and ERK1 /2 in H9c2 cells. Results Compared with the H / R group,S1 P significantly increased vaibility of cells,lowered the rate of apoptosis,decreased the content of MDA in the culture medium,increased the activity of T-SOD and Mn-SOD, reduced concentration of intracellular calcium and increased the phosphorylation of Akt and ERK1 /2. When added LY294002 or PD98059,the effects of S1 P above were inhibited. Conclusion S1 P protects H9c2 cells against hypoxia / reoxygenation injury. The protection of S1 P was inhibited by LY294002,the inhibitor of PI3 K / Akt and PD98059,the inhibitor of ERK1 /2. S1 P protects H9c2 cells against hypoxia /reoxygenation injury via RISK pathway.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2015年第2期181-185,共5页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81173058)
国家自然科学基金青年基金资助项目(No 81102446)
天津市自然科学基金资助项目(No 10JCZDJC20900)
